Phosphate deficiency alters transcript isoforms via alternative transcription start sites

Area of Science:

• Plant Molecular Biology
• Gene Regulation
• Plant Physiology

Background:

• Alternative transcription start sites (TSS) are common in eukaryotes, influencing mRNA 5' UTR length and protein-coding potential.
• Phytohormone treatments have shown effects on TSS usage, but the impact of nutrient availability, such as inorganic phosphate (Pi), is less understood.

Purpose of the Study:

• To investigate the role of inorganic phosphate (Pi) availability in regulating alternative transcription start site (TSS) usage in Arabidopsis thaliana.
• To characterize the downstream effects of Pi-regulated alternative TSS on gene expression, translation, and subcellular localization.

Main Methods:

• Combined short-read and long-read RNA sequencing to identify and analyze full-length mRNAs with alternative TSS.
• Bioinformatic analysis to identify genes with differential TSS usage under Pi deficiency.
• Functional analysis to assess the impact of alternative TSS on protein translation, subcellular localization, and root development.

Main Results:

• Identified 45 genes with altered TSS usage under Pi deficiency, with Pi having a specific and pronounced effect compared to phytohormones.
• Demonstrated that alternative TSS impact translation (e.g., upstream open reading frames in RLB1) and subcellular localization (e.g., plastid transit peptide in HO1 and SQDG2).
• Discovered a novel short noncoding transcript (ALT_ECT4) generated by an alternative TSS in the ECT4 locus under Pi deficiency, implicating m6A readers in Pi response and root meristem cell division.

Conclusions:

• Alternative TSS usage is a significant regulatory mechanism in the plant's response to inorganic phosphate (Pi) deficiency.
• Pi-regulated alternative TSS contribute to modulating gene function, including translation, localization, and the generation of noncoding RNAs.
• The study uncovers a novel role for m6A readers in mediating primary root growth under low Pi conditions, linked to iron metabolism and root meristem activity.